In the following discussion certain articles and methods will be described for background and introductory purposes. Nothing contained herein is to be construed as an “admission” of prior art. Applicant expressly reserves the right to demonstrate, where appropriate, that the articles and methods referenced herein do not constitute prior art under the applicable statutory provisions.
Rapid advances in DNA sequencing have created a pressing need for new technologies that enable the translation of genomic sequence information into information about protein function at the level of the proteome. Proteomics, the study of the function, structure and interaction of proteins, requires the ability to produce and study proteins in a high throughput manner. Traditionally, one approach has been to use combinatorial chemical synthesis methods to make large collections of peptides. However, these methods provide a random sampling of all possible n-mers, and are therefore inefficient for generating compact collections of protein sequences that are enriched for sequences of high biological relevance, such as peptides representing the human proteome. Highly specific and sensitive high-throughput methods for assaying proteins as a large collection are also lacking. Protein microarrays are a useful tool for such high throughput analysis of proteins, but the availability of microarray technology for large scale proteomics studies is still very limited due to the difficulty and cost of protein production (see Henderson and Bradley, Curr. Opin. Biotechnol., 18(4):326-30 (2007), Epub 2007 Aug. 6; and Tapia, Methods Mol. Biol., 570:3-17 (2009)).
Traditionally, peptide arrays are made by spotting pre-synthesized peptides on a surface (Salisbury, et al, J. Am. Chem. Soc. 124(50):14868-70 (2002)) or by synthesizing peptides in spots on cellulose filter sheets using standard solid phase peptide synthesis, also known as the SPOT method (Frank, J. Immunol. Methods, 267(1):13-26 (2002)). However, the cost of generating arrays with tens of thousands or more spotted peptides is very high. This is a major impediment to the use of large arrays of peptides for most applications, and severely limits accessibility of large arrays to researchers. Several methods enable direct chemical synthesis of peptides in microarray format, which reduces costs, but these methods still have the major drawback of variability in the quality of the synthesized peptides (Antohe and Cooley, Methods Mol. Biol., 381:299-312 (2007)). Moreover, the direct fabrication process can be very slow and inefficient (Hilpert, et al., Nat. Protoc., 2:1333-49 (2007)).
Recently, methods for peptide array fabrication by in vitro translation have been developed, including protein in situ array (PISA) production (He and Taussig, Nucleic Acids Res., 29: e73 (2001)), nucleic acid programmable protein array (NAPPA) production (Ranachandran, et al., Science, 305:86-90 (2004)), DNA to protein array (DAPA) construction (He, Nat. Methods, 5:175-177 (2008), and arraying of proteins using in situ puromycin capture (Tao and Zhu, Nat. Biotech, 24:1253-1254 (2006)).
These approaches require individually synthesized nucleic acid templates, however, and the cost of these templates is higher than the cost of individual peptides arrayed by traditional methods. In addition, analysis of the peptides is limited to substrate-based systems.
The ability to manufacture large, high-quality, sequence-diverse peptide sets in solution, coupled with labeling methods and techniques compatible with high-throughput analysis of such large peptide sets, would enable high-throughput binding and enzymatic activity profiling studies having important applications in research, diagnostics and therapeutic development. The present invention addresses this need.